The long-term goal of the proposed research is to understand the role of molecular motors in organizing signaling pathways relevant to human disease. This study will investigate the intracellular trafficking of amyloid precursor protein (APP), the precursor of amyloid beta peptide, which forms the extracellular senile plaques in Alzheimer's disease. Recent data suggest that APP is transported to the cell surface by kinesin-I, where it may regulate cell movement, and that its proteolytic cleavage product, Cgamma is transported retrogradely (presumably by cytoplasmic dynein), and functions in signaling to the nucleus. The anterograde transport of APP and the retrograde transport of Cgamma are highly regulated processes, and may occur in conjunction with the stress-activated, c-Jun NH2-terminal kinase (JNK) signaling complex, and the scaffolding protein, Fe65 that may be involved in the pathogenesis of Alzheimer's disease. This regulation will be studied in CAD cells (a catecholaminergic cell line derived from the central nervous system), which show features of both normal neurons, and of degenerating neurons present in the brains of Alzheimer's disease patients. The first SpecificAim will address the role of phosphorylation ofAPP by the kinases, Cdk5 and JNK in regulating recruitmentof kinesin-I to APP and, thereby transport of APP into axons. Real-time imaging will be used to analyze the motility of fluorescently-taggedAPP in conditions that do or do not block its phosphorylation. To determine the role of Cdk5 and JNK inAPP transport, motility will be analyzed in conditions in which their activity is suppressed, and correlated with data from biochemical experiments that test the interactionof APP with kinesin-I. By using a similar methodology, as well as subcellular fractionation and ultrastructural localization, the second Specific Aim will investigate whether APP and another kinesin-I cargo, the JNK signaling complex, are transported independently (i.e., on separate vesicles), or together, as part of the same cargo. The third Specific Aim will address the participation of kinesin-I and cytoplasmic dynein in the transport of signaling complexes that containAPP or Cgamma, and the scaffolding protein, Fe65. Information gained throughout the study will be used to reconstitute APP/JIP-1 and APP/Fe65 motility in vitro. This study should reveal key insights into a possible cross-talk between signaling pathways involving APP, JNK, and Cdk5, with implications for brain development and neurodegeneration.